Microdevice for cell separation utilizing activation phenotype

ABSTRACT

Disclosed is a system and method for a microdevice to separate blood cells based on differences in antigen expression. Specifically, cells of the same phenotype are separated based on whether or not they are activated during infection or resting. The device of the present disclosure takes a small sample of blood and provides differential cell counts that can be used to test for infection and inflammatory response. The device can be used to identify sepsis and other infections rapidly. By measuring differences in activated white cell counts such as neutrophils, the device of the present disclosure measures physiological response to infection in hospitalized patients recovering from burns, surgeries, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase filing of PCT/US2015/054979, filedon Oct. 9. 2015, entitled “Microdevice For Cell Separation UtilizingActivation Phenotype” which claims priority to provisional U.S. PatentApplication Ser. No. 62/061,739, filed on Oct. 9, 2014, entitled“Microdevice for Cell Separation Utilizing Activation Phenotype” whichprovisional patent application is commonly assigned to the Assignee ofthe present invention and is hereby incorporated herein by reference inits entirety for all purposes.

This application includes material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent disclosure, as it appears in thePatent and Trademark Office files or records, but otherwise reserves allcopyright rights whatsoever.

TECHNICAL FIELD

The present disclosure relates in general to the field of diagnosis anddetection of biological samples. In particular, the system provides fora microdevice to separate blood cells based on differences in antigenexpression. The disclosed systems and methods support a wide variety ofscenarios and includes various products and services. Examples ofend-use applications include the detection of sepsis, isolation ofactivated neutrophil cells, and the detection of proliferating cellsfrom resting cells.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

BACKGROUND OF THE DISCLOSURE

Infection is defined as the invasion of a host organism's body tissuesby disease-causing agents, their multiplication, and the reaction ofhost tissues to these organisms and the toxins they produce. Infectiousdiseases, also known as transmissible diseases or communicable diseases,comprise clinically evident illness (i.e., characteristic medical signsand/or symptoms of disease) resulting from the infection, presence andgrowth of pathogenic biological agents in an individual host organism.

Infections are caused by infectious agents such as viruses, viroids, andprions, microorganisms such as bacteria, nematodes such as roundwormsand pinworms, arthropods such as ticks, mites, fleas, and lice, fungisuch as ringworm, and other macroparasites such as tapeworms.

Hosts can fight infections using their immune system. Mammalian hostsreact to infections with an innate response, often involvinginflammation, followed by an adaptive response.

Sepsis is a potentially fatal whole-body inflammation (a systemicinflammatory response syndrome or SIRS) caused by severe infection.Sepsis is caused by the immune system's response to a serious infection,most commonly bacteria, but also fungi, viruses, and parasites in theblood, urinary tract, lungs, skin, or other tissues. Sepsis can also becaused by toxins even when no identifiable bacteria are present. Sepsiscan be thought of as falling within a continuum from infection tomultiple organ dysfunction syndrome.

Documenting the presence of the pathogenic microorganisms that areclinically significant to sepsis has proven difficult. Causativemicroorganisms typically are detected by culturing a subject's blood,sputum, urine, wound secretion, in-dwelling line catheter surfaces, etc.Causative microorganisms, however, may reside only in certain bodymicroenvironments such that the particular material that is cultured maynot contain the contaminating microorganisms. Detection may becomplicated further by low numbers of microorganisms at the site ofinfection. Low numbers of pathogens in blood present a particularproblem for diagnosing sepsis by culturing blood. In one study, forexample, positive culture results were obtained in only 17% of subjectspresenting clinical manifestations of sepsis (Rangel-Frausto et al.,1995, JAMA 273:117-123). Diagnosis can be further complicated bycontamination of samples by non-pathogenic microorganisms.

Common symptoms of sepsis include those related to a specific infection,but usually accompanied by high fevers, hot, flushed skin, elevatedheart rate, hyperventilation, altered mental status, swelling, and lowblood pressure. In the very young and elderly, or in people withweakened immune systems, the pattern of symptoms may be atypical, withhypothermia and without an easily localizable infection.

In addition to symptoms related to the provoking infection, sepsis isfrequently associated with fever or hypothermia, rapid breathing,elevated heart rate, confusion, and edema. Early signs are elevatedheart rate, decreased urination, and elevated blood sugar, while signsof established sepsis are confusion, metabolic acidosis withcompensatory respiratory alkalosis (which can manifest as fasterbreathing), low blood pressure, decreased systemic vascular resistance,higher cardiac output, and dysfunctions of blood coagulation.

Prompt diagnosis is crucial to the management of sepsis, as initiationof early-goal-directed therapy is key to reducing mortality from severesepsis. Within the first three hours of suspected sepsis, diagnosticstudies should include measurement of serum lactate, obtainingappropriate cultures before initiation of antimicrobial treatment, solong as this does not delay antimicrobial treatment by more than 45minutes. To identify the causative organism(s), at least two sets ofblood cultures (aerobic and anaerobic bottles) should be obtained, withat least one drawn percutaneously and one drawn through each vascularaccess device (such as an IV catheter) in place more than 48 hours. Ifother sources are suspected, cultures of these sources, such as urine,cerebrospinal fluid, wounds, or respiratory secretions, should beobtained as well, so long as this does not delay antimicrobialtreatment.

Within six hours, if there is persistent hypotension despite initialfluid resuscitation of 30 ml/kg, or if initial lactate is ≧4 mmol/L (36mg/dL), central venous pressure and central venous oxygen saturationshould be measured. Lactate should be re-measured if the initial lactatewas elevated.

Within twelve hours, it is essential to diagnose or exclude any sourceof infection that would require emergent source control, such asnecrotizing soft tissue infection, peritonitis, cholangitis, intestinalinfarction. Sepsis may also lead to a drop in blood pressure, resultingin shock. This may result in light-headedness. Bruising or intensebleeding may also occur. The aforementioned, gross parameters have notbeen identified as specific to sepsis.

Sepsis is usually treated with intravenous fluids and antibiotics. Iffluid replacement is not sufficient to maintain blood pressure,vasopressors can be used. Mechanical ventilation and dialysis may beneeded to support the function of the lungs and kidneys, respectively.To guide therapy, a central venous catheter and an arterial catheter maybe placed; measurement of other hemodynamic variables (such as cardiacoutput, mixed venous oxygen saturation or stroke volume variation) mayalso be used.

Infections cause millions of deaths globally each year. Sepsis and otherinfections are usually characterized by blood culture, which takes 24-48hours to identify. Currently, the mortality rate for sepsis is high dueto this long waiting period.

Identifying sepsis and other infections earlier can increase survivalrates by over 10 times. However, there are still limitations for currentdetection methods and devices.

SUMMARY OF THE DISCLOSURE

The present disclosure addresses failings in the art by providing asystem and method for rapid detection of infections, such as sepsis.

The present disclosure provides for a microdevice to separate bloodcells based on differences in antigen expression. Specifically, cells ofthe same phenotype are separated based on whether or not they areactivated during infection or resting. The device of the presentdisclosure takes a small sample of blood and provides differential cellcounts that can be used to test for infection and inflammatory response.The device can be used to identify sepsis and other infections rapidly(less than 12 hours and as low as 6 hours). By measuring differences inactivated white cell counts such as neutrophils, the device of thepresent disclosure measures physiological response to infection inhospitalized patients recovering from burns, surgeries, etc.

The present disclosure identifies sepsis as soon as six hours afterinfection, allowing physicians to directly measure the body's responseto infection. Such information allows physicians and other careproviders to respond rapidly to patients with massive inflammatoryresponse to an infection, and thus decrease mortality. The device of thepresent disclosure may be further implemented in any clinical settingdue to its ease of use and low cost. The device of the presentdisclosure achieves 97-99% accuracy for cell identification in testedblood cell separation from control samples.

It is an object of the present disclosure to provide differential cellcounts in a short-time span in order to reduce patient mortality in thedetection and/or diagnosis of certain infections.

It is a further object of the present disclosure to provide for themeasurement of physiological response to infections in patients.Patients may range from those experiencing acute symptoms of infectionto those recovering from burns or surgeries, or other post-incidentalinfection management and control.

It is a further object of the present disclosure to allow for the quickidentification of infections, particularly sepsis (a condition in whichtimely diagnosis is of the essence). In some embodiments, the system ofthe present disclosure may be implementable in any clinical setting dueto ease of use and low cost. The technology can be utilized to measuredifferential cell counts of blood. Further, the present disclosureprovides for a diagnostic test for infections and inflammationsutilizing a microfluidic device of varying arrangements to separate andmeasure the presence of activated cells, such as neutrophils.

It is yet another object of the present invention to provide amicrofluidic detection chip for the detection of infection in a patientcomprising: a plurality of layers in which are disposed a plurality ofchannels; a sample input channel into which a sample fluid mixture ofcomponents to be isolated is inputted; one or more separation channelshaving one or more three-dimensional (3D) separation zones; and one ormore channels having one or more optical zones. The one or more 3Dseparation zones further comprise at least one vertical interface and atleast one horizontal interface. The chip may further comprise aseparation channel for monocyte depletion. The one or more separationchannels may be arranged serially or in parallel. In one aspect, the oneor more separation channels comprises an affinity surface comprising abiotinylated antibody, such as anti-CD4, anti-CD19 and the like. Thechip may conform to known chip configurations, and may be at least threelayers. The separation channels may further comprise a first affinitysurface and a second affinity surface for capturing varying levels ofexpressive cells, such as high-expression neutrophils in a firstaffinity surface, and a second affinity surface which captures restingneutrophils. Further, the one or more separation channels may comprise afirst affinity surface and a second affinity surface arranged in seriesor in parallel channels. Once captured, it is an object of the presentinvention to provide enumeration of cells by cell imaging, which mayoccur by flatbed scanning, which may further comprise using contrastingagents.

It is another object of the present invention to provide a method ofidentifying the presence of infection, comprising: flowing a patientsample through a microfluidic device having a substrate having formedtherein one or more separation channels, at least one portion of the oneor more separation channels having a plurality of monolayers, wherein atleast a portion of said monolayers comprises a monocyte affinity surfacecapable of cell capture, and one or more optical zones having aplurality of monolayers, wherein at least a portion of said monolayerscomprises a neutrophil affinity surface capable of cell capture;capturing active neutrophils in at least one separation channel;capturing resting neutrophils in at least one separation channel;enumerating the active and resting neutrophils in the optical zone; anddetermining the ratio of active-to-resting neutrophils. The method mayfurther include depleting monocytes from the sample in the separationchannels prior to neutrophil capture. The plurality of separationchannels may have more than one monocyte affinity surfaces capable ofcell capture of more than one monocytes and may further comprise 3Dchannels.

In one aspect, the monocyte affinity surface comprises a biotinylatedantibody. In addition, the neutrophil affinity surface comprises atleast one surface having a biotinylated antibody. In another aspect, themethod includes enumerating the active and resting neutrophils using anoptical scanner. The optical scanner may be a flatbed scanner and mayfurther utilize a high-contrast dye in bright field. Fluorescencescanning is also possible with the present invention.

It is an object of the present invention to provide separation designthat results in enhanced cell capture in affinity microchannels. Thishigh capture efficiency and capture purity allows for removal ofunwanted cells from a sample (negative selection) before isolated targetcells (positive election). The affinity chips can isolate cell typesthat cannot be isolated in a single-step analysis. In addition, methodsare provided to remove monocytes prior to analysis, a requirement foraccurate CD64+ neutrophil counting. A further aspect is to use a singlechip to isolate neutrophils based solely on whether they are resting(low CD64 expression) or active (high CD64 expression).

In accordance with one or more embodiments, a system is provided thatcomprises one or more microfluidic devices configured to providefunctionality in accordance with such embodiments. In accordance withone or more embodiments, functionality is embodied in steps of a methodperformed by at least one computing device. In accordance with one ormore embodiments, program code to implement functionality in accordancewith one or more such embodiments is embodied in, by and/or on acomputer-readable medium particularly with cell enumeration.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of thedisclosure will be apparent from the following description ofembodiments as illustrated in the accompanying drawings, in whichreference characters refer to the same parts throughout the variousviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating principles of the disclosure:

FIG. 1 depicts a microfluidic schematic, in parallel configuration, formeasuring the presence of levels of CD64 bodies within a blood sample.

FIG. 2 (A) depicts the three-dimensional (3D) separation channel fornegative depletion; and (B) a magnified view of enhanced cell capture ofthe 3D channels.

FIG. 3 depicts separation of neutrophils for sepsis assays using aserial channel.

FIG. 4 depicts saturation of the affinity surface as measured by cellspassing through the outlet of each 3D stage (y-axis represents cellsleaving chip, not retained in channel).

FIG. 5 depicts flow rate effects in cell capture.

FIG. 6 depicts antibody concentration (loaded into the chip) affects oncell capture.

FIG. 7 depicts two embodiments of a microdevice to separate blood cellsbased on differences in antigen expression (anti-CD64 antibody): (A)comprising a parallel chip orientation; and (B), comprising a seriesorientation.

FIG. 8 depicts herringbone channel capture efficiency as a function offlow rate in the chip (bottom).

FIG. 9 depicts isolation of CD4+(A) and CD19+(B) cells from lysed bloodused tandem cell affinity columns.

FIG. 10 depicts multi-parameter analysis of a cell mixture using 4serial affinity regions.

FIG. 11 depicts control of cell capture efficiency by controllingantibody concentration.

FIG. 12 depicts USAF 1951 Resolution Bar Target image obtained using adesktop flatbed scanner at 2400×2400 DPI resolution.

FIG. 13 depicts optimized optical scanning configurations for On-Chipreadouts.

DETAILED DESCRIPTION OF THE DISCLOSURE

While the making and using of various embodiments of the presentdisclosure are discussed in detail below, it should be appreciated thatthe present disclosure provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts, goods, orservices. The specific embodiments discussed herein are merelyillustrative of specific ways to make and use the disclosure and do notdelimit the scope of the disclosure.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this disclosure pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The present disclosure will now be described more fully hereinafter withreference to the accompanying drawings, which form a part hereof, andwhich show, by way of illustration, specific example embodiments.Subject matter may, however, be embodied in a variety of different formsand, therefore, covered or claimed subject matter is intended to beconstrued as not being limited to any example embodiments set forthherein; example embodiments are provided merely to be illustrative.Likewise, a reasonably broad scope for claimed or covered subject matteris intended. Among other things, for example, subject matter may beembodied as methods, devices, components, or systems. Accordingly,embodiments may, for example, take the form of hardware, software,firmware or any combination thereof (other than software per se). Thefollowing detailed description is, therefore, not intended to be takenin a limiting sense.

Throughout the specification and claims, terms may have nuanced meaningssuggested or implied in context beyond an explicitly stated meaning.Likewise, the phrase “in one embodiment” as used herein does notnecessarily refer to the same embodiment and the phrase “in anotherembodiment” as used herein does not necessarily refer to a differentembodiment. It is intended, for example, that claimed subject matterinclude combinations of example embodiments in whole or in part.

In general, terminology may be understood at least in part from usage incontext. For example, terms, such as “and”, “or”, or “and/or,” as usedherein may include a variety of meanings that may depend at least inpart upon the context in which such terms are used. Typically, “or” ifused to associate a list, such as A, B or C, is intended to mean A, B,and C, here used in the inclusive sense, as well as A, B or C, here usedin the exclusive sense. In addition, the term “one or more” as usedherein, depending at least in part upon context, may be used to describeany feature, structure, or characteristic in a singular sense or may beused to describe combinations of features, structures or characteristicsin a plural sense. Similarly, terms, such as “a,” “an,” or “the,” again,may be understood to convey a singular usage or to convey a pluralusage, depending at least in part upon context. In addition, the term“based on” may be understood as not necessarily intended to convey anexclusive set of factors and may, instead, allow for existence ofadditional factors not necessarily expressly described, again, dependingat least in part on context.

The development towards lab-on-a-chip devices has been greatly advancedsince the first introduction of microfluidics for chemical analysis,with much progress being made in the area of chemical separations. Theattractiveness of microfluidics-based separations to analytical chemistsowes to its ability to routinely perform rapid and sensitive experimentson a small footprint with minimal use of sample and reagents.Additionally, microfluidic technology facilitates the fabrication ofdevices with integrated functions an advantage which has led to amultitude of microdevices for pertinent biochemical assays. The successof microchips in basic research has led to the birth of specializedcommercial devices for multiple bioanalytical applications, includingapplications in cell biology, genetics, pharmacology, and otherbiomedical fields.

Sepsis, systemic inflammatory response syndrome (SIRS), and septic shockrepresent significant causes of hospitalization and death in the UnitedStates. Sepsis is the 10th highest cause of death with an associatedhealthcare cost of 16.7 billion dollars. Sepsis-related hospitalizationsare 75% longer than patients treated for other conditions. In addition,the percentage of hospitalizations with sepsis-related principlediagnoses has increased from 11.6% in 2000 to 24.0% in 2008. Currently,sepsis is assayed using blood culture or other devices that detect thepresence of bacteria. The timeline for detecting sepsis in this mannerranges from 24-48 hours. These blood culture methods do not detectsepsis or septic shock in cases where there are no detectable bacteria.

Sepsis and septic shock continue to be major problems in the treatmentof burn and surgery patients. The diagnosis of sepsis typically requiresblood culture, which can take 24-48 hours. The long analysis timerequired for blood culture increases the likelihood of mortality.Alternatively, the physiological responses associated with sepsis can beused as a diagnostic tool. It is known in the art that neutrophilactivation is particularly attractive as a quantification of sepsis,since some forms of sepsis have no detectable bacterial infection.Activation of neutrophils during sepsis and SIRS can be measured viachanges in CD64 expression and are detectable as early as 3-6 hoursafter infection. Neutrophil activation is typically measured usingfluorescence activated cell sorters and magnetic activated cell sorters;however, they are not readily implemented in every medical setting. Fora sepsis assay that can be translated to point of care settings, asimpler, automated approach is needed. Microfluidic methods based onaffinity cell chromatography can be adopted in research and clinicalsettings with minimal cost and infrastructure. While many approacheshave been developed for cell separations, most methods isolate cells ina single separation step. Activated and resting neutrophils havediffering levels of the same surface marker, making separating these twocell types and quantifying them difficult.

Separating and sorting cells from a heterogeneous mixture is afundamental step in basic biological, chemical, and clinical studies. Insepsis, a blood culture is typically conducted, requiring several daysbefore a positive result is obtained. Procalcitonin is also used toassay sepsis. However, procalcitonin is also present at elevated levelsin other diseases such as malaria and community-acquired pneumonia.However, it is possible to assay the biological response to sepsis,looking for neutrophil activation and subsequent increase in CD64expression. Cell separations can be used for a rapid assay of sepsis,enumerating activated CD64+ neutrophils from 0.1 mL blood samples in asimple measurement. Cell separations are typically performed using cellsorters or magnetic beads, although other approaches such asdielectrophoresis, magnetophoresis, filtration, and affinity methodshave been developed. Most cell isolation methods use one or moredifferent antigens for cell enrichment. However, in neutrophil isolationtwo groups of cells are isolated—resting and activated neutrophils—thatshare the same antigens, although they express them at different levels.The key challenge therefore is to separate high- and low-expressingCD64+ neutrophils from each other and from other blood types. Thepresent invention utilizes a chip with different regions of cell captureefficiency to isolate activated and resting neutrophils. Separatingcells based on differences in antigen expression requires differences inflow geometries, surface chemistry, or other factors. A chip that canmeasure differences in neutrophil activation further enables cliniciansto identify systemic response to injury or infection, and would aid insepsis treatment. In addition, automating the analysis and enumerationof cell capture could lead to point of care systems for sepsis assay—acritical need for burn or surgery patients.

It is therefore an embodiment of the present invention to provide cellseparation microfluidic chips for analysis of neutrophil activation.These devices will be developed toward producing simple, inexpensive,and rapid tests for CD64+ neutrophil activation counts for point of caresepsis assays. The present invention measures neutrophil activationusing affinity cell separations in a microfluidic format and is furtherable to measure resting and activated neutrophils from small samples ofblood. In one embodiment, affinity regions with different CD64+ cellcapture efficiencies will differentially isolate resting and activatedneutrophils, and that those cells can be enumerated for rapid andinexpensive sepsis assays. These assays are capable of detecting changesin CD64 expression in neutrophils within 3-6 hours of sepsis or SIRS,which is 3-8 times earlier than bacterial assays, wherein themeasurement of resting and activated neutrophils is allowed.

The present invention provides for separation systems that use a uniqueflow architecture to achieve high-efficiency cell separations. Thesystem utilizes three-dimensional (3D configurations, such asherringbone structures for efficient cell capture. This approach allowsfor increased cell-antibody interaction on the chip surface. Inaddition, the approach is easy to fabricate and is amenable to opticalscanning using simple instrumentation. Each 3D flow interface increasescell capture. Resting and activated neutrophils can be separated usingeither parallel channels or using serial affinity regions in the samechannel. In one embodiment of the present invention, there are twoserial affinity regions, each corresponding to cell capture for activeor resting neutrophils. A serial affinity design eliminates differencesin flow between microfluidic channels and can be used to isolatedifferent cell types from blood. In both series and parallel chips, ananti-CD64 affinity region with low capture affinity will let restingneutrophils pass while capturing activated neutrophils having higherexpression of CD64. A second anti-CD64 affinity region with high captureaffinity will then capture resting neutrophils. The entire chip is thenscanned on a flatbed scanner and cell counting is performedautomatically in software to provide the ratio of activated and restingneutrophils.

Cell separations take advantage of physical differences between cellssuch as size and density, or differences between antigen expression onthe cell surface. Dielectrophoresis, sedimentation, and filtrationapproaches have been developed to isolate cells based on differences inphysical properties. In many applications where cell separations arenecessary, differences in physical parameters alone are not sufficientto isolate the desired cell type with high purity. Approaches thatexploit differences in antigen expression or other affinity-typeinteractions include fluorescence activated cell sorting (FACS),microfluidics FACS, magnetic activated cell sorting, and affinitymethods. However, affinity separations, including FACS, cannot easilyisolate cells when more than one cell type expresses the antigen that isrecognized by the affinity ligand. It is possible to use multipleaffinity tags in FACS, but the cost and complexity of existinginstruments preclude point of care diagnosis in most cases. Microfluidicmethods for separating similar cell types have not separated identicalcell types with different antigen expression levels.

Sepsis, SIRS, and septic shock are traditionally diagnosed initiallywith gross symptoms such as low blood pressure, elevated heart rate(>90/min), elevated respiratory rate (>20/min), elevated or decreasedwhite cell count (>12,000 or <4,000 cells/mm3), and fever (>38° C.).These methods have a wide degree of variability and can be subjective.Sepsis is then further diagnosed using blood culture although newermethods have been developed to detect bacterial contamination in blood.These approaches still require sufficient time for bacterial levels torise to their respective detection limits. Typical detection times are24-48 hours after infection, which coincides with the typical mortalityrate for patients with septic shock. The present invention detectsphysical response to infection, which manifests itself more rapidly.Procalcitonin assays are typically measured after 24 hours, making themless suitable for early detection of sepsis.

The present invention utilizes multiple separation zones are used underboth positive and negative selection to isolate cells that cannot easilybe separated by other methods. Unlike 96-well formats, the chips of thepresent invention isolate cells of interest based on affinityseparation, followed by whole-chip scanning for enumeration. Negativeselection affinity regions can remove interfering cells prior toanalysis by positive selection. For example, monocytes (which expressCD64) may interfere with neutrophil enumeration. It therefore an objectof the present invention to deplete monocytes prior to the blood samplereaching the anti-CD64 affinity regions for neutrophil capture. In theserial affinity chip after monocyte depletion, the first affinity zonecaptures neutrophils with high CD64 expression, thus depleting thosecells prior to capture in the second affinity zone. The second affinityzone therefore captures resting neutrophils. In parallel channel chipsof the present invention, blood is split between two channels, one thatcaptures all neutrophils with high affinity and one channel thatcaptures activated neutrophils only. Both approaches will be evaluatedto determine the best approach for activated neutrophils detection. Itis another embodiment of the present invention to provide theincorporation of on-chip enumeration. In an exemplary embodiment, usinga flatbed scanner and a contrast dye added to the running buffer cellsare imaged. Flatbed scanners have been used for cell imaging in partbecause of the large field of view as well as the ability to usecommercially available, low-cost systems. Readout can occur usingfluorescence or contrasting agents. The approach will automaticallycount cells to further speed up diagnostics. It is an exemplaryembodiment to utilize contrasting agents for flatbed scanning usingdesktop scanners.

The separation chips of the present invention are therefore designed toincrease capture efficiency, capture purity, and separation speed.Microfluidic affinity cell separations require efficient cell-surfaceinteractions to retain cells in the separation channel. It is shown thatsuch affinity approach isolates target cells from blood with 97-99%purity, and that cell capture is greatly enhanced when cell-affinitysurface interaction increases. It is thus another embodiment of thepresent invention to provide enhanced capture surfaces for greaterinteraction between the cells and the capture ligand to enhance captureefficiency and purity. In an exemplary embodiment, a herringbone mixerwill increase capture efficiencies higher than extant approaches. Theincreased capture surface interaction results in high captureefficiencies that approach 100%. The 3D chip approach is thereforeutilized to deplete unwanted cells (e.g. monocytes) prior to isolationof neutrophils using imaging-friendly herringbone channels. Usingdifferent flow geometries and affinity capture strategies, separationregions are created capable of isolating activated and restingneutrophils, respectively, thus improving capture efficiency overpillar- and herringbone-type separations alone, enabling cell analysesthat have been difficult or impossible in the past by providing chiparchitecture that will use affinity ligands for efficient cell capturefor sepsis assay studies.

Neutrophilic granulocytes express their Fcgamma receptor I, also knownas the CD64 antigen, predominantly when they are activated. This makesneutrophil CD64 a known biomarker for infection and sepsis. Indeed thereis ample literature on the diagnostic utility of neutrophil CD64 in avariety of diseases. IgG responses are crucial in the diagnosis ofinfections. Assessing their activation in vitro is of fundamentalimportance, but technically difficult.

CD64 is a type of integral membrane glycoprotein known as an Fc receptorthat binds monomeric IgG-type antibodies with high affinity. It is morecommonly known as Fc-gamma receptor 1 (FcγRI). After binding IgG, CD64interacts with an accessory chain known as the common γ chain (γ chain),which possesses an ITAM motif that is necessary for triggering cellularactivation. Human CD64 is a high affinity receptor for monomeric humanIgG1 and IgG3 which is expressed on macrophages, monocytes, and gammainterferon induced neutrophils. CD64 plays an important role inclearance of immune complexes and in antibody dependent cytoxicity.

In the present invention, CD64 antibodies, having locus BC032634 andknown as Homo sapiens Fc fragment of IgG, high affinity Ia, receptor(CD64), mRNA (cDNA clone MGC:45021 IMAGE:5248549), complete cds,Accession BC032634, Version BC032634.1 GI:21619685, are utilized in amicrofluidic channel capable of contacting detectably the CD64antibodies specific for CD64 with blood leucocytes in a sample obtainedfrom the subject. The resulting measurable CD64-antibody complex isidentified as abnormal expressions, and suggests the presence ofinfection.

The principles discussed herein may be embodied in many different forms.The preferred embodiments of the present disclosure will now bedescribed where for completeness, reference should be made at least tothe Figures.

FIG. 1 shows an exemplary detection chip 100 of the present invention. Asample 101 is introduced into an inlet having one or more channels forseparation 102. The sample 101 may be a blood sample ranging from100-1000 μL. The channels 102 allow for the sample to flow across thechannels which are layered with anti-CD64 bodies. This may be controlledby a control valve (not shown) or by drawing via an outlet 103. FIG. 1shows the application of the present disclosure to the detection anddiagnosis of SIRS, or sepsis, wherein a CD64-affinity chip is utilized.The chip, oriented as a parallel chip, utilizes CD64 antibody todistinguish active and resting states of CD64, a known marker.Enumeration of the distinguished active neutrophils and thedistinguished resting neutrophils, present an opportunity to calculateand therefore diagnose, sepsis. The enumeration may occur at an opticalchannel 105, wherein optical enumeration is achieved by scanning usingcell counting beams via laser or LED.

Several approaches have been developed to increase the interactionbetween cells and the affinity surface. Under most microfluidicconditions, flow is laminar and only cells near the channel surface willinteract with affinity ligands. Both micropillars and herringbone mixershave been used to isolate cells either in positive or negative selectionmodes. However, neither reported approach has been able to achieve highcapture efficiencies, which are required for depletion of unwanted celltypes for affinity separations. In one embodiment of the presentinvention, deceleration at the interface between vertical and horizontalchannels, a so-called 3D separation stage, increases the interactiontime between the cell and the affinity surface. FIG. 2(A) presents anexemplary 3D herringbone configuration 201. Fluorescence correlationspectroscopy (FCS) measurements of single molecules flowing through thechip showed a marked deceleration at each vertical interface, increasingcell retention. The chips reached separation purities of 93%. Anexemplary embodiment of the present invention is in the use of multiple3D stages to overcome the problem of cell surface overloading. Thedownward trajectory approach to cell separations results in captureefficiencies that overload the affinity surface with cells. As cellsoccupy the entire capture surface, target cells will pass through theaffinity region without being captured. However, in the presentinvention, each additional 3D stage presents a new affinity surface. Aseach 3D stage is overloaded sequentially, the remaining stages remainfunctional to ensure a large number of cells can be captured. FIG. 2(B)provides a more detailed view of a separation 3D channel used fornegative depletion before neutrophil capture, which can exceed 98%purity with high capture density 202. In this case CD4+ cells (HuT 78cell line) were captured with high efficiency. Turning to FIG. 4 it isshown that the number of cells passing through the first 3D stage(Outlet 1) increases after 20 minutes. Saturation of the affinitysurface as measured by cells passing through the outlet of each 3D stage(y-axis represents cells leaving chip, not retained in channel). CD4+HuT 78 cells were depleted from a cell mixture using an anti-CD4 chipwith 6 3D interfaces. After 20 minutes, the first 3D affinity surfacewas completely covered with target cells. At that point, both target andbackground cells could pass through the channel without capture,degrading separation performance. Using multiple 3D interfaces in ourchip ensures that as one affinity surface is saturated, downstreamsurfaces are available for cell capture. The downward trajectory of asingle 3D interface ensures high cell capture, requiring multipleinterfaces to avoid saturation. At this point in time the affinitysurface was saturated with cells and no additional capture could occur.If only one interface was used, then separation performance woulddecrease at that time. Using multiple 3D stages increases the totalcapture capacity. Since the 3D design is not amenable to facile on-chipimaging, this channel type will only be used to isolate unwanted cellsto deplete interference cells before CD64 neutrophil capture, whichoccurs via one or more separation channels.

In early experiments, it was shown that cell capture using the downwardtrajectory approach was 12 times greater than a straight channel ofsimilar dimensions and linear flow rates. While typical chip designs mayhave high capture capacity, purity, and efficiency, the latter can befurther increased using additional microfluidic structures. In anotherembodiment, chips are designed with several affinity regions placed inseries. These chips deplete and capture one blood cell type in eachregion, and can be used to isolate leukocytes from blood with highpurity (see Table 1). In this case, an anti-CD4 region preceded theanti-CD19 region, and both regions captured their respective cell typeswith >97% purity. These chips use straight affinity sections referred toherein as separation channels, resulting in a 50-60% capture efficiency.However, both the 3D chip approach and the serial affinity sections canbe combined for high purity and high efficiency cell separations.

TABLE 1 Blood Cell Separations Using Affinity-sectioned Chips, Li 2012.Anti-CD4 Region Anti-CD19 Region Target Cells/mL 360 ± 40 130 ± 70 TotalCells/mL 370 ± 30 130 ± 70 Lot-certified Cells/mL 707 (CD4+) 214 (CD19+)Capture Purity (%) 99.1 ± 0.4 97 ± 2 Capture Efficiency (%) 51 61 Li,2012

FIG. 3 presents an exemplary embodiment of the principles of theseparation channel conveyed herein. Chips 300 are made usingpoly(dimethylsiloxane) (PDMS) bonded to glass slides. Fluid flow 305 iscontrolled by syringe pumps or automated means known in the art, andlight and fluorescence microscopy is used to assess separations in situ.The chip 300 presents the configuration of separation of neutrophils forsepsis assays using a serial channel. The first separation zone 301 hasfewer antibodies on the surface, resulting in lower capture efficiency.Neutrophils showing increased antigen expression are captured in thefirst separation zone 301 while neutrophils with low antigen expressionpass to the second zone 303 and are captured on a high-efficiencycapture surface. Flow cytometry verifies cell mixtures before and afterseparations as a control measurement. A first separation channel 301contains a low antibody concentration. A second separation channel 303comprises a high antibody concentration. A control valve is provided 302for flow modulation if desired. The separation channels 301 and 303 haveherringbone channels for providing the 3D interfaces for cell capture ofexpressing cells 306, 307. Non-expressing cells are then capable offlowing out of the separation channels 301, 303. Separation channels forthe purposes of the present invention may also be utilized for monocytedepletion, as well as for concurrent enumeration of the captured cells,which separation channel may be referred to as an optical channel.

The chips of the present invention may be three-layer devices includinglayers for control lines. The affinity surface can be glass or PDMS. Theexemplary designs have a first section with six, repeating 3D interfacesper affinity section for CD14 capture. The dimensions of each interfacewill first be modeled using COMSOL software to optimize cell-surfaceinteractions. COMSOL further assists in modeling the number ofinterfaces needed to capture cells with the highest efficiency.Validations of the modeling results occurs using anti-CD71 captureantibodies and Ramos cells to determine the cell capture with theCOMSOL-optimized geometries as a starting point.

Chip-to-chip variability is further determined in cell capture and flowrate. Flow rate may be assessed by visually tracking cell movementthrough the chip via microscopy. Variations in fabrication can causechanges in the linear flow rate when the same volumetric flow rate isused. In affinity cell separations a cell is captured on the surface ifthe total affinity adhesion force exceeds the shear force. The number ofbonds (B_(c)) needed to retain a cell on the affinity surface can beexpressed as B_(c)=F/f_(c), where F is the shear force and f_(c) is thesum of the adhesion force from all affinity bonds between the cell andthe surface. The number of bonds formed during a cell-surfaceinteraction (B*) is expressed as:

B*=t _(c) A _(c) B,

where B is the density of bonds formed per unit area, t_(c) is theduration of interaction, and A_(c) is the contact area between the celland the affinity surface. Faster flow rates result in less cell capture(see FIG. 5) and the B*/B_(c) ratio results in cell capture atvalues >1. This ratio is inversely proportional to the square of thevolumetric flow rate. Increasing the volumetric flow rate decreases thecell interaction time, t_(c), (decreasing B*) and increases the shearforce, F (increasing B_(c)). This 1/x² dependence requires carefulcontrol of the flow rate. As shown in FIG. 5 there is a rapid drop offin cell purity that approaches the initial concentration ratio of thetarget cells. Cells were isolated from a mixture by negative selection.The initial ratio of target cells was 53% (dashed line). Slower flowrates resulted in higher separation purity, as background cells werebetter retained in the chip. To assess flow rate variability betweenchips, cell suspensions are flowed through the chips at the samevolumetric flow rate and measure linear flow rate of cells by videomicroscopy. Anti-CD19 surfaces are used with Ramos B cells as the targetcell. Ramos cells are then mixed with CCRF-CEM cells (which are CD19−)to determine capture purity (Ramos Cells vs. total cells captured),nonspecific binding (CCRF-CEM cells vs. total cells captured) andcapture efficiency. To differentiate between the two cell types, Ramoscells are incubated with MitoTracker Green and CCRF-CEM cells withHoechst 33342 and fluorescence microscopy is then used to count cells.The cell samples and concentrations remain the same for all chip-to-chipvariability studies. Captured cells in the chip are measured todetermine if the small changes in flow rate affect cell capture. Thevolumetric flow rates are then adjusted in the same test chips todetermine if the same linear flow rate affects differences in cellcapture, informing the inter-chip variation in flow and cell capture,and for correcting for differences in flow rate decreases variability.

The overall design of the sepsis chip of the present invention involvestwo separation regions using anti-CD64 antibodies. The two sectionsdiffer by capture efficiency. Capture efficiency can be modulated usingflow effects (FIG. 5), antibody concentration (FIG. 6), or by usingdifferent types of separation channels to achieve different captureefficiencies. In FIG. 6 antibody concentration (loaded into the chip)shows effects on cell capture. As expected, at high concentrations (>20μg/mL) the effect of antibody concentration on cell capture decreases.At lower antibody concentrations, the relationship between cell captureand antigen expression is linear (inset), allowing differences inantigen expression to be measured using the chip There may also existthe need to deplete monocytes from the sample prior to CD64 capture(FIG. 7). To maintain high capture efficiency but also aid in opticalscanning, herringbone-modified chips used to capture neutrophils aftermonocyte depletion. Herringbone mixers have been used in cellseparations to induce chaotic mixing in the microchannel, increasinginteraction between cells and the capture surface. Herringbone sectionshave high capture efficiency (FIG. 8) and it is demonstrated thatantigen expression can be elucidated using these chips. The herringbonechips have greater capture efficiency at higher flow rates than normal,straight channels. For the present invention herringbone regions areused after the 3D depletion section chips as an alternate strategy toincrease capture efficiency.

FIG. 7 provides two alternating orientations of the system of thepresent invention for separating active and resting neutrophils. In FIG.7A monocytes are depleted (optional) from the blood sample, which isthen split into two streams of differing CD64 capture efficiency. Acontrol valve allows captured neutrophils to be eluted for automatedcounting. FIG. 7B, the two anti-CD64 channels are operated in series(detection occurs after each section). The monocyte depletion sections(first separation region) can be either a 3D chip, herringbone, orcombination of the two. The FIG. 7(A) orientation 701 provides aparallel chip orientation wherein both resting 703 and active 704 cellsare detecting utilizing a simultaneous exposure to antibody samples forthe alternative channels. An inlet 712 introduces the sample through aseparation channel 705 which comprises an anti-CD14 surface. A controlvalve 702 further allows for flow modulation if desired. On the parallelchip 701, active neutrophils 704 and resting neutrophils 703 are exposedto antibody samples, such as anti-CD64, allowing for capture andenumeration. The FIG. 7(B) orientation shows a series-based chip 706,providing for the antibodies to be exposed to both active 709 andresting 710 cell states in serial fashion rather than simultaneously. Aninlet 712 introduces a sample into the separation channel 707 whichcomprises an anti-CD14 surface. A control valve 708 further allows forflow modulation if desired. The series configuration passes the samplethrough a first zone 709 capable of removing active neutrophils, whilethe second zone 710 provides for capture of resting neutrophils. Witheither configuration 701, 706 the enumeration of the captured andresting neutrophils will provide determination of the presence ofinfection.

With previous work in cell separations, CD4+ and CD19+ cells wereisolated from blood using two open-tubular affinity columns connected inseries (see FIG. 9). The purity of CD4+ leukocytes was 87% with 0.2% ofcaptured cells CD19+. The purity of the CD19+ lymphocytes was 82% with0.1% of the captured cells measured as CD4+. It is therefore possible touse a series approach to remove one cell type before the second celltype is separated. Therefore in one embodiment, depletion zones removemonocytes prior to CD64+ neutrophil isolation. This approach is capableof capturing multiple cell types in a chip, as shown in FIG. 10. In FIG.10, a chip with four separation regions in series was used on a mixtureof HuT 78, Ramos, and CRL-1435 cell lines. The chip contained multipleaffinity regions using valves to control surface coating. As expected,the anti-CD4 region captured HuT 78 cells with a small degree ofnonspecific binding, while the anti-CD19 region captured Ramos cellswith no nonspecific binding. All three of the cell lines express CD71and were captured on the anti-CD71 region. An anti-CD8 region was usedto evaluate nonspecific binding in this case, as none of the cell linesexpress CD8.

Additionally, cell capture may be modulated by varying the antibodyconcentrations on a chip. In FIG. 11, the effect of antibodyconcentration in a serial herringbone separation chip is shown with twoaffinity regions. Control of cell capture efficiency by controllingantibody concentration. A herringbone chip was used to capture CD71+Ramos cells spiked into blood. The Ramos cell CD71 expression rangedbetween 3-40 times higher than CD71+ leukocytes in blood. The captureratio of Ramos cells to CD71+ leukocytes was measured in the chip, witha linear relationship between the cell capture and the antigenexpression (y=0.95X−0.11, R2=0.95). As the CD71+ expression increased inRamos cells (conducted on different days with the same blood samples)the ratio of Ramos:Leukocytes in the chip increased linearly. Theseresults demonstrate that the chips can measure changes in antigenexpression and are capable of processing complex samples, such as blood.The first region has a lower concentration of anti-CD19 to serve as alow-efficiency capture region and the second region has a higheranti-CD19 concentration. The ratio of cell capture was calculated as thecell counts in the second region (high efficiency capture) divided bythe cell counts in the lowest antibody concentration capture region. Itwas therefore possible to control the cell capture by changing theantibody concentrations at a single flow rate. This approach simplifieschip design and operation. The range of antigen expression between twocell lines capture in the device was 3- to 40-fold. Since it is expectedfor neutrophil CD64 expression to increase 10-20× during sepsis, thepresent invention is then able to generate two affinity regions capableof separating active and resting neutrophils.

In another exemplary embodiment of the present invention, an affinitysurface is generated using sandwich approach commonly used for cellseparations. The surface is first coated with biotinylated BSA, followedby a layer of neutravidin. A biotinylated antibody is then added tocomplete the surface coating. This approach allows any biotinylatedcapture molecule to be used for chip separations. These surfaces arestable under refrigerated, dry storage, and can be functionalized withthe final antibody layer in minutes before use, or stored with theantibody for weeks at 4° C.

Cell separations may be conducted using either stop-flow orcontinuous-flow strategies. In stop flow, the chip is filled with lysedor whole blood, and cells are allowed to settle to the surface forcapture. A wash step then removes unbound cells and enumeration occurs.In continuous flow, cells are introduced at a flow rate that ensures lowshear force (in the exemplary chips of the present invention, typically0.05 mL/hr). Sample is continuously introduced in this case, and higherflow rates are used to wash unbound cells prior to cell enumeration.Stop flow methods typically have higher purity, although lower totalsample numbers. However, since the objective is capturing neutrophils,with typical concentration ranges of 2,500-6,000 cells/mL, stop flowmethods will result in a sufficient number of cells. The absoluteneutrophil count is less important than the active/resting neutrophilratio. In exemplary embodiments, the volume of a single 3D interfacesection may be around 0.15 mL. Given the range of neutrophils in blood,the number of cells that can be injected into that volume is 2,200-5,400cells (per 3D section). If the capture efficiency is 55% (an average ofprevious results, Table 1), then it is expected that 1,100-2,700 cellsper section, or 6600-16,000 CD64+ neutrophils for the entire CD64isolation channel in stop flow mode. In the case of measuring active vs.resting neutrophils, this would represent the total cell count, with thenumbers of resting and active cells varying depending on the state ofthe donor. The number of cells isolated in stop flow will therefore besufficient for statistical analysis of cell counts. However, if the cellcounts are insufficient in stop flow mode, continuous flow can be usedto introduce larger total cell volumes. A 20-minute separation wouldrequire 30 mL of blood and would inject 75,000-180,000 neutrophils intothe chip. Cell lysis requires 1.5 minutes, and washing steps are notnecessary. The dye used for image contrast is included in the runningbuffer, and the scan time to read the entire chip (or several chips atonce) is on the order of 30 seconds. The entire analysis time, fromsample to answer, is therefore less than 25 minutes. This analysis timeis comparable with the staining time when using antibodies and flowcytometry or magnetic separations for CD64 neutrophil counting.

Once modeling reveals the best initial geometries, high-efficiencychannels can be used for both the capture of CD64+ resting neutrophilsand also to deplete monocytes from blood prior to neutrophil analysis.In both cases, the highest cell capture possible is desired. Since thecell mixture plays a role in the reproducibility, inter-chip variationstudies are further conducted with the same sample mixture on the sameday.

The lower-efficiency anti-CD64 region will be optimized to captureneutrophils that are activated and have higher CD64 expression. Inaddition to herringbone channels, chip efficiency may be altered tocapture resting and active neutrophils differently. In one embodiment anunmodified channel with an anti-CD64 surface captures activatedneutrophils, while a herringbone approach is needed to capture restingneutrophils. In another embodiment the flow rate of the chip is alteredto increase or decrease capture efficiency. One way to alter the flowrate is to change the channel dimensions, but this approach may alsochange capture efficiency in unforeseen ways. In an exemplaryembodiment, in order to optimize chip designs, commercially availablebeads are utilized with known differences in antigen density. Theseantigen-density beads are commonly used in flow cytometry as standardsto measure antigen expression. Beads with antigen densities are usedmatching the densities of CD64 on activated and resting neutrophils.This approach allows chip designs to be optimized in a controlledsystem. In cell capture chips, the height of the channels is critical,and the length (along the flow direction) is also important. Of lessimpact on capture performance is the width of the channel, and thisdimension can be reduced to increase the linear flow rate of this chipsection. Using this approach, faster moving cells will have lessinteraction time and cell capture efficiency will decrease. The channelwidth may be optimized so that >90% capture of activated high antigendensity beads is observed while minimizing capture of lower antigendensity beads to <10%. Given the differences in CD64 expression betweenactivated and resting neutrophils, this difference in capture efficiencyis feasible. Cells that exit the narrow anti-CD64 section will thenenter another anti-CD64 that has channels that are similar in dimensionto the anti-CD14 region. The restoration of a slower linear flow ratewill increase capture efficiency so that lower antigen density cells orbeads are captured.

The above embodiments may exist as alternate approaches or incombination with the multiple fluidic architectures presented forefficient cell separations. While there may be preferable singleapproach, it is also possible that the present invention combinesdifferent channel designs in the same chip.

In addressing sepsis models, in one embodiment the present invention isdesigned to deplete monocytes prior to CD64+ neutrophil using bloodsamples. Commercial sources of blood are utilized to validate monocytedepletion and neutrophil capture. Determined are the necessary flowrates, sample volumes, and analysis times as well as antibody coatingrequired for neutrophil capture. Currently flow cytometry is thestandard method for such an analysis, using several fluorescent antibodylabels to identify cells. However, any newly developed methods thatcould simplify such analysis and reduce the cost of measurement would beapplicable on sepsis assays for point of care diagnosis. Affinitymethods to date have run into interferences from cells that also expressCD64, such as monocytes. It is possible to use magnetic beads ordifferential shear force to remove cells, however this approach isimprecise. In one embodiment, first-stage cell separation unitscomprising a plurality of separation channels, which may be 3D, or acombination of herringbone mixers or micropillars, and the like, areutilized to deplete interfering cells so that a subsequent separationstage can isolate target cells for analysis. This approach is based ontandem separations. The first zone will operate under the principle ofnegative selection, capturing monocytes and preventing them fromentering subsequent separation regions.

In an exemplary embodiment of the present invention regarding bloodsample preparation, standardized blood samples available from commercialsources are not identified in any way or linked to donors. The benefitof using such blood samples is that they are analyzed beforehand andhave certified concentrations of all major blood cell types. CD14+monocyte depletion in lysed and whole blood is then tested. Lysisprotocols are implemented using deionized water or NH₄Cl-based buffers,followed by a saline buffer to restore proper salinity to leukocytes. Toassess the influence of erythrocytes on our chip separations, chips ofthe present invention are coated with anti-CD14 to deplete monocytes.The same blood sample is utilized for all chip designs, which compareCD14+ monocyte concentrations in the chip effluent using flow cytometryand fluorescence microscopy. Monocytes are stained with anti-CD14 AlexaFluor 647 for identification. By measuring cells that pass through thechip, the capture efficiency in whole and lysed blood is determined. Forlysed blood, leukocytes are centrifuged and re-suspended at the sameconcentration as in the original blood, in order to eliminateconcentration effects on cell capture. In this manner, the effects oferythrocytes are compared to blood viscosity to lysed blood samples oncell capture. It is anticipated that if whole blood does not yieldsufficient performance (>90% capture efficiency of CD14+ lymphocytes),then lysed blood would be used in future protocols.

To assess monocyte depletion, chips with only one separation sectioncoated with anti-CD14 are used. The effluent of these chips will beanalyzed to determine cell depletion. For exemplary purposes, both thecapture efficiency of monocytes as well as the capture capacity aremeasured. The total number of monocytes anticipated in a standard sampleof blood (30 mL) is on the order of 8000 cells in the chip. Each 3Dsection for monocyte depletion can capture a theoretical maximum of 2200cells, based on surface coverage, which can approach 100% (see FIG. 2).Therefore six 3D sections in series captures all monocytes in the bloodsample. The capacity for monocyte measurement does not need to beexcessively high, but the capture efficiency should be high and thenonspecific binding low. CD64+/CD14+ monocyte concentrations beforeseparation and after elution from the anti-CD14 are measured, as well ascapture capacity by monitoring the monocyte population eluting from thechip over time. A sharp increase in the rate of monocytes exiting thechip will signal that the affinity surface is saturated (see FIG. 4).The separation channel is also monitored using light microscopy in realtime, to observe cell capture effects. In designing the chip, the mostimportant criteria will be the capture efficiency. However, the rate ofnonspecific binding must also be low. CD64+ neutrophil capture should bepreferably 0.1% or less.

One issue with blood measurements of any kind is the presence oferythrocytes at higher numbers than leukocytes. However, a lysisprotocol can be used to preserve leukocytes while removing erythrocytesprior to analysis. It is shown repeatedly in literature that theremaining fragments of erythrocytes do not impede cell separation. Theother issue is nonspecific binding in the CD14 and CD64 capturechannels. The present invention is capable of separating blood cellswith 97-99% purity, and with enhanced separation efficiency the accuracythus improves.

In another embodiment of the present invention, the chip configurationeliminates the need to manually count cells or image them viamicroscopy. The cells are counted in the chip after the sample has beenintroduced, using flatbed scanner technology generally available. Inorder to count cells in the chip, a contrast dye is added to the runningbuffer that will stain all cells for bright field or fluorescenceimaging. Labeling all cells has an inherently high signal-to-noise ratiowhen compared to immunofluorescence. The automated system will aid intranslation of the present invention to the clinical lab use. Opticalscanning using a flatbed scanner allows for microfluidic chips to beimaged inexpensively in a user-friendly format. Optical scannerresolution for an inexpensive ($250-$300) desktop scanner often exceeds2400×2400 dots per inch (DPI), with higher resolution possible usinginterpolative scanning. The preliminary image in FIG. 12 shows a USAFresolution target scanned with a 2400×2400 DPI desktop scanner. Thesystem resolves features that are 16 mm apart (2400×2400 interpolation).Given the scale of cells imaged in the system of the present invention(10-15 mm), modern scanning systems are able to resolve cells in thechip. The 5,1 element was resolved, yielding a spatial resolution of 16μm. This preliminary result shows that an inexpensive scanning systemhas spatial resolution approaching single cells.

FIG. 13 shows the scanning, or enumeration, action of the presentinvention. For the present invention two approaches that can be used foroptical scanning of cells, depending on the dye used to stain cells forcontrast. In FIG. 13A, a dark-contrast dye stains all cells 1303 and thechip is read out using reflectance as the scanner 1300 progresses 1301across the separation channel (referred to also as an optical channel).The scanner head 1300 provides the light source and detector in thiscase. This approach requires minimal/no hardware modification. Aspresented herein, a desktop scanner approaches single cell resolution.In FIG. 13B, a bank of LEDs 1307 (only 1 is shown for clarity)illuminates the chip from above. All cells 1304 in this case are stainedwith a fluorescent dye such as Hoechst 33342 and the chip is imaged1305. A thin film 1306 under the chip blocks excitation light, so thatonly fluorescence light is measured. The cells can be counted individualor an aggregate signal can be measured. If bright field imaging is used,it is feasible to utilize the standard reflected light optical scanningapproach that is traditionally used in flatbed scanners. This approachrequires no hardware modification. The benefit of using this approach isthat any scanner with sufficient resolution and contrast may be used. Inaddition, since the scan area of most scanners is 8×11″ or 11×17″multiple chips may be imaged at once if needed. For fluorescenceimaging, an array of LEDs is placed above the chip to illuminate thedevice uniformly. A dye stains all cells and fluorescence is imagedthrough a thin film filter to block the LED illumination. This approachhas higher signal to noise than reflectance imaging. The LED module isconfigured to house a chip, and can then be placed on any suitablescanner for imaging. For exemplary purposes, the spatial resolution andcontrast is evaluated using USAF 1951 Resolution Bar Targets and USAFContrast targets for both approaches.

In an exemplary embodiment, the contrast dye will be added to therunning buffer during the separation. Hoechst 33342 is an example dyethat will stain all cell nuclei for counting, but will not contributesignificant background fluorescence. For bright field imaging, Nile Bluederivatives can be detected either by bright field or fluorescence. Tooptimize and calibrate our system, cells of known concentration areloaded into the chip and counted via standard microscopy. Counting maythen be performed with IMAGEJ software and a custom plugin. Actual cellcounts are compared with the aggregated signal (i.e. total fluorescenceor dye), to see which approach is most accurate. The latter will reducethe optical resolution required, but may be less accurate than countingcells directly. The software tabulates the total cell counts from eachaffinity region and will provide the ratio of cells counted in theactive and resting neutrophil regions as well as the total neutrophilcount. It is expected that there will be some cross-talk between CD64+neutrophils, but that the ratio of cells in the two regions willcorrelate with sepsis.

In addressing sensitivity and spatial resolution of optical scanning ofthe entire chip, single-cell resolution is possible by using ahigh-contrast dye in bright field or fluorescence scanning to eliminatethe need for high sensitivity, resulting in a simple cell enumerationmethod that requires no user intervention. Once cells are loaded andseparated in the chip, the chip is placed on a flatbed scanner and cellsare counted as the entire chip is scanned. The automated enumerationsystem will help translate the present invention to point of caresystems.

Neutrophils have shown an increase in CD64 expression during sepsis.Increased CD64 expression is a direct result of infection, unlike othermethods that look at bacterial load. A burn, which increases totalneutrophil numbers, will not result in increased CD64 expression unlessinfection occurs. Therefore CD64 expression can be used as a diagnosticmarker for sepsis. Using an anti-CD71 capture channel, a ratio ofantigen density between two cell types is equivalent to the ratio ofcell capture in the same chip under identical conditions. It is then anembodiment of the present invention to generate channels where cellcapture efficiency is modulated by altering antibody concentration onthe chip surface. The antibody concentration may be kept constant butgenerate chip sections with different linear flow rates or capturegeometries if needed.

Pursuing optimized chip designs prevents the drawbacks of traditionallab-on-a-chip devices. Those drawbacks include subjective analysis andheavy operator burden (analysis time, functionality of devices, etc.).The automated approach of the present invention will decrease user errorand operator burden, and will allow for translation to point of carediagnosis. The cost per device is significantly lower than extantmethods such as flow cytometry and procalcitonin assays, and can beimplemented in a wider variety of clinical settings. Further, thepresent invention overcomes problems associated with current cellseparation methods, and result in isolation of target cells with highpurity. In one embodiment, the present invention provides a neutrophilassay as a relevant marker and assay for sepsis. Using several animalmodels to control the type of sepsis, the degree of sepsis and septicshock is assayed, allowing for setting quantitative cutoff values topredict sepsis.

It is therefore an embodiment of the present disclosure to provide asystem and method for utilizing a microdevice for the rapid detection ofan inflammatory response associated with infection which includes theblood stream. In another embodiment the detection rapidly determines thepresence of systemic inflammatory response syndrome, or SIRS, also knownas sepsis. The present invention includes an in-vitro, diagnostic, pointof care device, including a microfluidic channel for conducting patientblood samples for purposes of scanning and/or detection of levels ofdesired contents, or markers. In one embodiment, the system utilizescell counts to test for infection and inflammatory response, determiningwhether the cells of the same phenotype are separated based on whetherthey are ‘active’ or ‘inactive’.

In a preferred embodiment of the present disclosure, a microdeviceseparates blood cells obtained from a patient based on differences inantigen expression. Specifically, cells of the same phenotype areseparated based on whether or not they are activated during infection orresting. The device of the present disclosure takes a small sample ofblood and provides differential cell counts that can be used to test forinfection and inflammatory response.

Sepsis and other infection is usually characterized by blood culture,which takes 24-48 hours to identify. Currently, the mortality rate forsepsis is high due to this long waiting period. Identifying sepsis andother infections earlier can increase survival rates by over 10 times.It is another embodiment of the present disclosure to detect thepresence of infection in a rapid manner (less than 24 hours). In anotherembodiment, the presence of infection in the blood sample is determinedin less than 12 hours. In yet another embodiment, the presence ofinfection in the blood sample is determined in less than 8 hours. In yetanother embodiment, the presence of infection in the blood sample isdetermined in less than 6 hours.

In one embodiment a method for determining whether a subject has aninfection comprises contacting detectably labeled antibodies specificfor CD64 with blood leucocytes in a sample obtained from a patient;forming a measurable amount of CD64-antibody complex; identifyingabnormal expression of CD64 on blood leucocytes in a sample obtainedfrom the patient from the amount of CD64-antibody complex; andidentifying abnormal expression of the antibody complexes on the bloodleucocytes in the sample CD64-antibody complex, wherein abnormalexpression of CD64 is indicative of the subject having a bacterialinfection. In one embodiment the sample may be whole blood. In anotherembodiment, the leucocytes are neutrophils. The complexes are thenmeasured to determine the presence of active CD64 antibody complexes.Counting can be implemented using optical scatter, fluorescencedetection, electric resistance, electrical impedance, or other means ofregistering cells on the surface such as wide field bright- ordark-field imaging.

Those skilled in the art will recognize that the methods and systems ofthe present disclosure may be implemented in many manners and as suchare not to be limited by the foregoing exemplary embodiments andexamples. In other words, functional elements being performed by singleor multiple components, in various combinations of hardware and softwareor firmware, and individual functions, may be distributed among varioussoftware applications at either the client level or server level orboth. In this regard, any number of the features of the differentembodiments described herein may be combined into single or multipleembodiments, and alternate embodiments having fewer than, or more than,all of the features described herein are possible.

Functionality may also be, in whole or in part, distributed amongmultiple components, in manners now known or to become known. Thus,myriad software/hardware/firmware combinations are possible in achievingthe functions, features, interfaces and preferences described herein.Moreover, the scope of the present disclosure covers conventionallyknown manners for carrying out the described features as well as thosevariations and modifications that may be made to the hardware orsoftware or firmware components described herein as would be understoodby those skilled in the art now and hereafter.

Furthermore, the embodiments of methods presented and described asdiagrams, schematics or flowcharts in this disclosure (such as theFigures) are provided by way of example in order to provide a morecomplete understanding of the technology. The disclosed methods are notlimited to the operations and logical flow presented herein. Alternativeembodiments are contemplated in which the order of the variousoperations is altered and in which sub-operations described as beingpart of a larger operation are performed independently.

While various embodiments have been described for purposes of thisdisclosure, such embodiments should not be deemed to limit the teachingof this disclosure to those embodiments. Various changes andmodifications may be made to the elements and operations described aboveto obtain a result that remains within the scope of the systems andprocesses described in this disclosure.

REFERENCES  [1] Patent U.S. Pat. No. 7,767,395 B2  [2] Patent U.S. Pat.No. 8,439,835 B1  [3] Patent U.S. Pat. No. 8,518,648 B2  [4] Patent U.S.Pat. No. 5,804,370 A  [5] Patent App US 20110059858 A1  [6] Patent U.S.Pat. No. 8,669,113 B2  [7] Patent U.S. Pat. No. 8,304,230 B2  [8] PatentU.S. Pat. No. 8,476,028 B2  [9] Patent U.S. Pat. No. 7,645,573 B2 [10]Patent App US 20080114576 A1

What is claimed is:
 1. A microfluidic detection chip for the detection of infection in a patient comprising: a. a plurality of layers in which are disposed a plurality of channels; b. a sample input channel into which a sample fluid mixture of components to be isolated is inputted; c. one or more separation channels having one or more three-dimensional (3D) separation zones; and d. one or more channels having one or more optical zones.
 2. The microfluidic detection chip of claim 1, wherein the infection is sepsis.
 3. The microfluidic detection chip of claim 1, wherein the one or more 3D separation zones further comprise at least one vertical interface and at least one horizontal interface.
 4. The microfluidic detection chip of claim 3, further comprising a separation channel for monocyte depletion.
 5. The microfluidic detection chip of claim 1, wherein the one or more separation channels are arranged serially.
 6. The microfluidic detection chip of claim 1, wherein the one or more separation channels comprises an affinity surface comprising a biotinylated antibody.
 7. The microfluidic detection chip of claim 6, wherein the one or more separation channels are coated with anti-CD4.
 8. The microfluidic detection chip of claim 6, wherein the one or more separation channels are coated with anti-CD19.
 9. The microfluidic detection chip of claim 6, wherein the one or more separation channels are coated with anti-CD64.
 10. The microfluidic detection chip of claim 1, wherein the one or more separation channels comprises a first affinity surface and a second affinity surface.
 11. The microfluidic detection chip of claim 10, wherein the second affinity surface captures resting neutrophils.
 12. The microfluidic detection chip of claim 10, wherein the one or more separation channels comprises a first affinity surface and a second affinity surface arranged in series.
 13. The microfluidic detection chip of claim 10, wherein the one or more separation channels comprises a first affinity surface and a second affinity surface arranged in parallel channels.
 14. The microfluidic detection chip of claim 1, further comprising enumeration of cells by cell imaging.
 15. The microfluidic detection chip of claim 14, wherein the cell imaging is by flatbed scanning.
 16. The microfluidic detection chip of claim 14, wherein the cell imaging further comprises using contrasting agents.
 17. A method of identifying the presence of infection, comprising: a. flowing a patient sample through a microfluidic device having a substrate having formed therein one or more separation channels, at least one portion of the one or more separation channels having a plurality of monolayers, wherein at least a portion of said monolayers comprises a monocyte affinity surface capable of cell capture, and one or more optical zones having a plurality of monolayers, wherein at least a portion of said monolayers comprises a neutrophil affinity surface capable of cell capture; b. capturing active neutrophils in at least one separation channel; c. capturing resting neutrophils in at least one separation channel; d. enumerating the active and resting neutrophils in the optical zone; and e. determining the ratio of active-to-resting neutrophils.
 18. The method of claim 17, further comprising depleting monocytes from the sample in the separation channels prior to neutrophil capture.
 19. The method of claim 17, wherein the plurality of separation channels have more than one monocyte affinity surfaces capable of cell capture of more than one monocytes.
 20. The method of claim 17, wherein the plurality of separation channels further comprise 3D channels.
 21. The method of claim 17, wherein the monocyte affinity surface comprises a biotinylated antibody.
 22. The method of claim 17, wherein the neutrophil affinity surface comprises at least one surface having a biotinylated antibody.
 23. The method of claim 17, further comprising enumerating the active and resting neutrophils using an optical scanner.
 24. The method of claim 22, wherein the optical scanner is a flatbed scanner.
 25. The method of claim 22, further utilizing a high-contrast dye in bright field.
 26. The method of claim 22, further utilizing fluorescence scanning.
 27. The method of claim 17, wherein the infection is sepsis. 